[Not Applicable]
This invention relates to the field of cell culture and to the treatment of endocrine disorders. In particular, this invention pertains to methods of inducing endocrine differentiation in cell culture thereby providing endocrine cell suitable for transplantation into a host organism
Many kinds of cells can be grown in culture, provided that suitable nutrients and other conditions for growth are supplied. Thus, since 1907 when Harrison noticed that nerve tissue explanted from frog embryos into dishes under clotted frog lymph developed axonal processes, scientists have made copious use of cultured tissues and cells from a variety of sources. Such cultures have been used to study genetic, physiological, and other phenomena, as well as to manufacture certain macromolecules using various fermentation techniques known in the art. In studies of mammalian cell biology, cell cultures derived from lymph nodes, muscle, connective tissue, kidney, dermis and other tissue sources have been used.
Generally speaking, the tissue sources that have been most susceptible to the preparation of cell cultures for studies are derivatives of the ancestor mesodermal cells of early development. Tissues that are the progeny of the ancestor endodermal and ectodermal cells have only in recent years become amenable to cell culture, of a limited sort only. The cell types derived from the endoderm and ectoderm of early development include epidermis, hair, nails, brain, nervous system, inner lining of the digestive tract, various glands, and others. Essentially, long-term cultures of normal differentiated glandular and epithelial cells, particularly those from humans, are still not available.
In the instance of the mammalian pancreas, until the present invention, no scientist has had the opportunity of studying, and no physician has had the prospect of using for treatment, a cell culture of pancreatic endocrine cells that exhibited sustained cell division and the glandular properties typical of the pancreas.
Similar to neurons, the endocrine cells of the mammalian pancreas have been considered to be post-mitotic, i.e., terminal, essentially non-dividing cells. Recent work has shown that the cells of the mammalian pancreas (including those of humans) are capable of survival in culture, however, propagation of differentiated (mature) cells having endocrine function has met with, at best, limited success.
The inability to study pancreatic endocrine cells in culture has impeded the ability of medical science to progress in the area of pancreatic disorders. Such disorders include diabetes mellitus, a disease that impairs or destroys the ability of the beta cells of the islets of Langerhans (structures within the pancreas) to produce sufficient quantities of the hormone insulin, a hormone that serves to prevent accumulation of sugar in the bloodstream. Type I diabetes mellitus (insulin dependent, or juvenile-onset diabetes) typically requires full hormone replacement therapy. In a second (and more common) form of the disease, type II diabetes (sometimes referred to as late onset, or senile diabetes), treatment often does not require insulin injections because a patient suffering with Type II diabetes may be able to control his/her blood sugar levels by carefully controlling food intake. However, as many as 30% of these patients also have reduced beta cell function and therefore are candidates for hormone replacement therapy as well. Diabetes is not confined to humans, but has been noted in other mammals as well, such as dogs and horses.
The etiology of the diabetic disease condition is not fully understood. However, it has been noted that autoimmunity antibodies (antibodies that xe2x80x9cmistakenlyxe2x80x9d attack bodily structures) and/or certain T lymphocytes may have an involvement long before clinical symptoms of diabetes emerge. Evidence in this direction relies, in part, on successful treatment of recently diagnosed diabetic patients with cyclosporin, an immunosuppressive drug. Such treatment has been shown to prevent or cause remission of insulin-dependent diabetes mellitus in mice (Mori et al. (1986) Diabetologia 29:244-247), rats (Jaworski et al. (1986) Diabetes Res. 3:1-6), and humans (Feutren et al. (1986) Lancet, 11:119-123). A clinical test to detect the presence of these humoral and cellular immunoreactions would allow the screening of individuals in a pre-diabetic state, which individuals could then be prophylactically treated with immunosuppressive drugs.
Current treatment of individuals with clinical manifestation of diabetes attempts to emulate the role of the pancreatic beta cells in a non-diabetic individual. Individuals with normal beta cell function have tight regulation of the amount of insulin secreted into their bloodstream. This regulation is due to a feed-back mechanism that resides in the beta cells that ordinarily prevents surges of blood sugar outside of the normal limits. Unless blood sugar is controlled properly, dangerous, even fatal, levels can result. Hence, treatment of a diabetic individual involves the use of injected bovine, porcine, or cloned human insulin on a daily basis.
Injected insulin and diet regulation permit survival and in many cases a good quality of life for years after onset of the disease. However, there is often a gradual decline in the health of diabetics that has been attributed to damage to the vascular system due to the inevitable surges (both high and low) in the concentration of glucose in the blood of diabetic patients. In short, diabetics treated with injected insulin cannot adjust their intake of carbohydrates and injection of insulin with sufficient precision of quantity and timing to prevent temporary surges of glucose outside of normal limits. These surges are believed to result in various vascular disorders that impair normal sight, kidney, and even ambulatory functions.
Both of these disease states, i.e., type I and type II diabetes, involving millions of people in the United States alone, preferably should be treated in a more regulated fashion. Successful transplants of whole isolated islets, for example, have been made in animals and in humans. However, long term resolution of diabetic symptoms has not yet been achieved by this method because of a lack of persistent functioning of the grafted islets in situ (see Robertson (1992) New England J. Med., 327:1861-1863).
For the grafts accomplished thus far in humans, one or two donated pancreases per patient treated are required. Unfortunately only some 6000 donated human pancreases become available in the United States in a year, and many of these are needed for whole pancreas organ transplants (used when the pancreas has been removed, usually during cancer surgery). Therefore, of the millions of diabetic individuals who could benefit from such grafts, only a relative handful of them may be treated given the current state of technology. If the supply of islet cells (including but not necessarily limited to beta cells) could be augmented by culturing the donated islets in cell culture, expanded populations would provide sufficient material to allow a new treatment for insulin-dependent diabetes.
This invention provides methods of culturing cells that differentiate and provide cells having endocrine activity in vitro. The methods generally involve culturing the cells in the presence of a phosphatidylinositol 3-kinase (PI3K) inhibitor. By using a PI3K inhibitors in the culture media, the ratio of endocrine positive (i.e. hormone producing and/or secreting cells) to endocrine negative cells is dramatically increased. Preferred mammalian cells include endocrine precursor cells, more preferably pancreas endocrine precursor cells (e.g. cells capable of differentiating into pancreas endocrine cells). Particularly preferred cells are pancreas cells (adult or fetal), more preferably human pancreas cells. Suitable phosphatidylinositol 3-kinase inhibitors include, but are not limited to wortmannin, a wortmannin analogue, Ly294002, and a Ly294002 analogue.
These culture methods thus provide a means by which large quantities of previously unavailable endocrine positive cells can be obtained. These cells find a number of uses, for example in the treatment of conditions characterized by a hormone deficiency (e.g. diabetes). Thus in another embodiment, this invention provides methods of treating a hormone deficiency in an organism, particularly a hormone deficiency characterized by a deficiency in insulin and/or glucagon, and/or somatostatin. The methods involve culturing a mammalian precursor cell in the presence of a phosphatidylinositol 3-kinase (PI3K) inhibitor whereby said precursor cell differentiates into a cell having endocrine activity; and then transplanting the cell having endocrine activity into said organism. The precursor cell can be virtually any endocrine precursor cell and more preferably is a pancreatic cell (e.g. a xcex2-cell). The method is particular well suited for treating conditions characterized by insulin deficiency (e.g. diabetes).
In another embodiment, this invention provides nutrient media suitable for the culture of differentiated mammalian cells having endocrine activity. In a preferred embodiment, the nutrient medium comprises a mammalian cell culture medium and a phosphatidylinositol 3-kinase inhibitor. Preferred inhibitors include, but are not limited to wortmannin, a wortmannin analogue, Ly294002, or a Ly294002 analogue and preferred culture media include, but are not limited to Eagle""s Basal Medium (BME), Eagle""s Minimum Essential Medium (MEM), Minimum Essential Medium with Non-Essential Amino Acids (MEM/NEAA), Dulbecco""s Modification of Eagle""s Medium (DMEM), McCoy""s 5 A, and Rosewell Park Memorial Institute (RPMI).
In still another embodiment, this invention provides a bioreactor. The bioreactor comprises a container containing a nutrient medium as described herein and a mammalian precursor cell capable of endocrine activity when differentiated.
Kits are also provided for the in vitro culture of differentiated endocrine cell(s). In a preferred embodiment, the kits comprise a container containing a phosphatidylinositol 3-kinase (PI3K) inhibitor and one or more other components selected from t5eh group consisting of a cell culture medium, adult mammalian cells, fetal mammalian cells, undifferentiated mammalian cells, partially differentiated mammalian cells, and/or instructional materials teaching the use of PI3K inhibitors to enhance the differentiation of endocrine cells in culture.
The following abbreviations are used herein: HGF/SF, hepatocyte growth factor/scatter factor; ICC, islet-like cell cluster; NIC, nicotinamide; PI3K, phosphatidylinositol 3-kinase; PI, phosphatidylinositol; PIP, phosphatidylinositol 4 phosphate; PIP2, phosphatidylinositol 4,5 bisphosphate; PIP3 or PtdIns(3,4,5)P3, phosphatidylinositol 3,4,5 trisphosphate; PKC, protein kinase C; PtdIns(3)P, phosphatidylinositol 3 phosphate; PtdIns(3,4)P2, phosphatidylinositol 3,4 bisphosphate.
The term xe2x80x9cdifferentiationxe2x80x9d refers to the process whereby cells or cell clones assume specialized functional biochemistries and/or morphologies previously absent. Such xe2x80x9cdeterminedxe2x80x9d cells may lose the ability to divide. Typically differentiation of a cell into one type of cell limits or prevents differentiation of a cell into another type. Differentiation of endocrine cells is characterized by the ability to express and/or secrete one or more hormones.
The term xe2x80x9cculture mediumxe2x80x9d, refers to a chemical composition that supports the growth and/or proliferation of a cell, preferably of a mammalian cell. Typical culture media include suitable nutrients (e.g. sugars, amino acids, proteins, and the like) to support the growth and/or proliferation of a cell. Media for the culture of mammalian cells are well known to those of skill in the art are include, but are not limited to Medium 199, Eagle""s Basal Medium (BME), Eagle""s Minimum Essential Medium (MEM), Minimum Essential Medium with Non-Essential Amino Acids (MEM/NEAA), Dulbecco""s Modification of Eagle""s Medium (DMEM), McCoy""s 5A, Rosewell Park Memorial Institute (RPMI) 1640, modified McCoy""s 5A, Ham""s F10 and F12, CMRL 1066 and CMRL 1969, Fisher""s medium, Glasgow Minimum Essential Medium (GMEM), Iscove""s Modified Dulbecco""s Medium (IMDM), Leibovitz""s L-15 Medium, McCoy""s 5A medium, S-MEM, NCTC-109, NCTC-135, Waymouth""s MB 752/1 medium, Williams"" Medium E, and the like. Cell culture media are commercially available (e.g. from GibcoBRL, Gaithersburg, Md.) and even custom-developed culture media are commercially available (see, e.g., Specialty Media, Cell and Molecular Technologies, Inc., Phillipsburg, N.J.).
The term xe2x80x9cendocrine activityxe2x80x9d as used herein refers to the activity of a cell in producing (expressing) and/or secreting a hormone (e.g. insulin, glucagon, etc.).
The terms xe2x80x9cprecursor cellxe2x80x9d or xe2x80x9cendocrine precursor cellxe2x80x9d as used herein refer to a cell that is capable of ultimately differentiating into a mature endocrine cell (e.g. a cell that produces (expresses) and/or secretes a hormone) under suitable conditions (in vitro and/or in vivo).
The term xe2x80x9cLy294002xe2x80x9d, as used herein, refers to the phosphatidylinositol 3-kinase inhibitor, 2-(4-Morpholinyl)-8-phenyl-4 H-1-benzopyran-4-one; as described by Vlahos, et al. (1994) J. Biol., Chem., 269(7) 5241-5248, and is available from Calbiochem Corp., La Jolla Calif.
Phosphatidylinositol (PI) 3xe2x80x2-kinase (Kazlauskas and Cooper (1989) Cell 58: 1121; Coughlin et al. (1989) Science 243, 1191) refers to a compound or compounds that phosphorylate the inositol ring of PI in the D-3 position (Whitman et al (1988) Nature 332, 644). PI3K activity is associated with a variety of activated tyrosine kinases and correlates with the presence of a tyrosine phosphorylated 85-kilodalton (kD) protein (p85) (Kaplan et al. (1987) Cell 50: 1021; Fukui and Hanafusa (1989) Mol. Cell. Biol. 9, 1651). Purified PI3K is a heterodimeric complex that contains p85 and a 110-Kd protein (p110) (Carpenter et al. (1990) J. Biol. Chem. 265, 19704). The purified p85 subunit has no detectable PI3K activity, but binds tightly to activated PDGFR or EGFR in vitro. PDGF stimulation induces accumulation of PI-3,4-P.sub.2 and PI-3,4,5-P.sub.3, confirming that PI3K is regulated by tyrosine kinases in vivo. Phosphatidylinositol-kinases belong, together with specific phospholipases, to an enzyme group which catalyses the formation of intracellular messenger substances from the membrane lipid phosphatidyl inositol (PI).